Insulators are elements required for transcriptional fidelity in eukaryotic genomes. Prevailing models suggest that insulators are structural components that divide chromosomes into loop domains to constrain enhancer and silencer action. The goal of our studies is to define mechanisms of insulator action, focusing on the Drosophila gypsy insulator of the gypsy retrovirus. This insulator is bound by the zinc finger protein, Suppressor of Hairy-wing [Su(Hw)], that recruits E(y)2 and two BTB domain proteins, Mod67.2 and CP190. Studies herein will elucidate mechanisms used by these proteins to regulate nuclear organization and gene expression in the Drosophila genome. A multi- faceted approach is proposed to understand the function of Su(Hw) at gypsy and non- gypsy sites in the genome. Three aims are proposed. First, we will determine the how the gypsy insulator blocks enhancer activated transcription. Second, we will define the role of insulator proteins in chromosome organization. Third, we will elucidate Su(Hw) function at non-gypsy sites in the female germline. These studies will provide new insights into models of insulator function and enhance our understanding of mechanisms involved in genome organization. PUBLIC HEALTH RELEVANCE: Insulators are genomic elements that define domains of independent transcription. Understanding mechanisms of insulator action is critical for improved design of gene therapy vectors, as current approaches face challenges such as inappropriate activation of cellular genes and inadequate long-term expression of therapeutic genes. Such studies will lead to the definition of discrete elements that can be included in the next generation of gene therapy vectors to enhance their effectiveness.